De-immunogenicity & Humanization 抗体去免疫原性 - 人源化. Le Sun, Ph.D. President & CEO AbMax Biotechnology Co, LTD

Transcription

1 De-immunogenicity & Humanization 抗体去免疫原性 - 人源化 Le Sun, Ph.D. President & CEO AbMax Biotechnology Co, LTD

2 抗体药市场发展迅猛

3 Top20 中的抗体药和主要 Players

4 抗体药面临的新挑战 临床研究及临床应用中显现的抗药性 化药 : DNA 突变 细胞水平 需要多年时间 不会直接致命 生物药 : 抗药抗体 ADA 免疫系统 仅需要几个月, 几个星期甚至几天 可能直接致死

5 免疫原性 ADA 风险巨大 5

6 阿达木病人治疗年失效率为 24% 6

7 多个抗体药因为 ADA 死于临床 3 期 给所有药企敲响警钟, 未来几年会有上百个抗体新药将因为 ADA 胎死腹中, 让投资人血本无归 Company Drug Indication Stage Abbvie/Roche Anti-IL2R 多发性硬化症 退市 Pfizer Anti-PCSK9 降血脂 Phase III Teva Anti-IL5 皮下注射治疗哮喘 Phase III AZ Anti-α4β1 肿瘤一线治疗 Phase III Genentech Anti-PD-L1 膀胱癌 肿瘤一线治疗 Phase III BMS Anti-PD1 肿瘤一线治疗 Phase III 研制出免疫原性低的创新型抗体药成为市场新的需求和未来抗体药发展的主要方向之一 Ridker et al. N Engl J Med. (2017) 376:

8 Sources of Immunogenicity-Internal ( 免疫原性来源 - 内源 ) Sequences: 1. Non-human sequences such as 鼠 兔和羊驼 2. Different genetic background such as HAHA (O>A/B>AB 基因背景 ) Modifications 1. 去糖基化 : Anti-PD-L1 Atezolizumab (41.8% ADA) 2. 去岩藻糖 (Afucosylation): 新的 B 细胞位点. 3. 纳米抗体 -Fc:Caplacizumab ( 需要换血和免疫抑制剂 ) 4. PEGylation: Anti-TNF Certolizumab pegol (35% ADA) 5. Antibody Drug Conjugation: Anti-HER2 Tadstuzumab emtansine (Herceptin 0.11%, T-DM1 5.3%) 6. Bi-specific: Anti-DLL4/Anti-VEGF Navicixzumab (33-36%) 8

9 研发误区 1- 为人源化而人源化 商品名 靶点 Type 适应症 免疫原性 Zevalin CD20 Chimeric NHL 2.50% Rituxan CD20 Chimeric NHL 11-23% Bexxar CD20 Chimeric NHL 10% Gazyva CD20 Humanized Chronic Lymphocytic Leukemia 7% 商品名 靶点 Type 适应症 免疫原性 Erbitux EGFR Chimeric Squamous Cell Head and Neck 5% Portrazza EGFR Human NSCLC) 4.10% Vectibix EGFR Human Metastatic Colorectal Cancer 5.30%

10 研发误区 2- 人 Germline 抗体就没有免疫原性 商品名 通用名 靶点 适应症 免疫原性 Yervoy Ipilimumab CTLA-4 Unresectable or Metastatic Melanoma 4.9% Portrazza Necitumumab EGFR NSCLC 4.1% Vectibix Panitumumab EGFR Metastatic Colorectal Cancer 5.3% Stelara Ustekinumab IL-12, IL-23 Psoriasis (Ps) 6.0% Praluent Alirocumab PCSK9 Primary Hyperlipidemia 4.8% Opdivo Nivolumab PD-1 Unresectable or Metastatic Melanoma 11.2% Humira Adalimumab TNFα Rheumatoid Arthritis (RA) 75.0% As immune responses against the variable regions of human mab are anticipated, FDA does not expect that the use of human mab will further reduce immunogenicity by a significant margin. 全人抗体照样有 ADA 问题

11 研发误区 3- 用 T 细胞表位去预测抗药抗体 T-cell Epitope Analysis Failed to Predict ADA The average affinities of peptides with E296V or M298Q mutation were significantly higher than peptides with V158D. K. Lamberth & Z. Sauna, 2017 Sci. Transl. Med. 11

12 T-cell Epitope Analysis Failed to Predict Humira s ADA 12

13 AbMax Speedy Ab developer mouse mab in 28 days, monkey pab in 35 days Made Abs against plants, cells, bacterial, virus, proteins, peptides, PnS, small molecules, fatty acids, etc Used mouse, rat, rabbit, goat, monkey, etc Anti-LRP5/6: 2002 J. Biol. Chem. Anti-PKHD1: 2004 Proc. Natl. Acad. Sci. Anti-PCDGF: 2004 Clin. Cancer Res. Anti-Wint5a: 2005 Nat. Genet. Anti-TNFR1 & 2: 2005 Nat. Med. Anti-phospho-PIP5K: : 2010 Immunity Anti-phospho-SSH2 : 2011 Dev Cell Anti-INMAP: 2013 Mol Cell Biochem Anti-EV71: 2013 Cytotechnology Anti-phospho-Axil: 2016 Dev Cell Anti-Metanzumab: 2016 J Immunol Methods Anti-Humira: 2018 J Immunol Methods Anti-DKK2: 2018 Nat. Med. 13

14 Antibody-Antigen Interaction Step 1 Docking: 3D Structures between Ab and Ag determine the specificity. Location visible or accessible 3D structure--outstanding Very often not species-dependent Step 2 Locking: H-bond and V-force at the contacting surfaces determine the affinity 14

15 AbEpiMax: 抗原表位最强化设计软件 Position within the protein Sequence homology Length: 10~18 a.a. Epitope score: hydrophilic, immunogenicity, etc Structure: rotation Non-antigenic: <0; Low: 0-7; High:8-15; Extremely high: >15 Designed >10,000 peptides as immunogens and made Abs against >5,000 targets 设计多肽去制备具有中和活性的抗体, 成功率高达 30%, 而其他软件不到 2%

16 天成的抗原位点分析准确地找到了产生 ADA 的 B 细胞表位 Mutation Antigenic Change V158D +5 E296V -4 M298Q +3.6 Triples +5 Target Ab Name Types Antigenic Score ADA rate in human PD-1 Nivolumab Fully human IgG % PD-1 Pembrolizumab Humanized IgG % TNF Adalimumab Fully human IgG % PCSK-9 Bococizumab Humanized IgG % 找到现有抗体药的 B 细胞位点并加以去除, 延长抗体药半衰期, 提高药物安全性

17 (1) lation of 2G1VH (1) n of 3F7VH1 rc (1) n of 4H8VH2 rc (1) tion of 5B3VH3 (1) tion of 5C5VH1 (1) n of 5F1VH1 rc (1) n of 5F8VH2 rc (1) Consensus (1) 重链轻链 Align VKLQQSGPGLVAPSQSLSITCTVSGFSLTSYDISWIRQSPGKGLEWLGVIWTG---GVTNYNSAFMSRLSISKDNSESQVFLKMTSLQTDDTAIYYCVRGGG---YPYSLDYWGQGTTVTVSS VKLQQSGAELVRPGASVKLSCKASGYSFTNYWMNWVKQRPGQGLEWIGMIHP--SDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCAREGRLGLRSYAMDYWGQGTTVTVSS VKLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMDNLRAEDTGIYYCTRRSS-----AWFAYWGQGTTVTVSS VKLQQSGPELVKPGASVRISCKASGYTFTIYYIHWVKQRPGQGLEWIGWIYP--GNVNAKYNEKFKGKATLTADRSSSTAYMQLNSLTSEDSAVYFCARSKG---SLYYFDYWGQGTTVTVSS VKLQQSGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGGINP--SNGGTNFNEKFKSKATLTVDKSSSTAYMQLNSLTSEDSAVYYCTIWS WFAYWGQGTTVTVSS VQLQQSGPGLVAPSQSLSITCTVSGFSLSTYGVHWVRQPPGKVLEWLGVIWAG---GSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCASG AWFAYWGQGTTVTVSS VKLQQSGPELVKPGASVRISCKASGYTFTIYYIHWVKQRPGQGLEWIGWIYP--GNVNAKYNEKFKGKATLTADKSSSTAYMQLSSLTSGDSAVYFCARSRG---SLYYFDYWGQGTTVTVSS VKLQQSGPELVKPGASVKISCKASGYTFT YYIHWVKQRPGQGLEWIG IWP N T YNEKFK KATLT DKSSSTAYMQL SLTSEDSAVYYCAR YWFDYWGQGTTVTVSS (46) ion of 2G1VK1 (1) DIQLTQSPAIMSASPGEKVTLTCSASSSVSSS-----YLYWYQQKPGSSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSFPYTFGGGTKLEIK of 3F7VK5 rc (1) DIQLTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQYPKLLVYFASTRESGVHDRFVGSGFGTDFTLTITSVQVEDLADYFCQQHYITPLTFGVGTKLEIK H8VK 自己拼接 (1) DIQLTQSPASLSMAIGEKVTIRCITSTDIDDD------MNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPCTRSEGGPSWRSN B3VK 自己拼接 (1) DIQLTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQ-YLSSYTFGGGTKLEIN ion of 5C5VK3 (1) DIQLTQSPASLAVSLGQRATISCQASESVSFAGT--SLMHWYQQKPGQPPKLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEDVHVRRGDQAGDL ion of 5F1VK2 (1) HSADPVSNPVTLGTSASISCRSSKSLLHN-NGITYLYWYLPKPGQSHHLLIYPMSNLASRIPDRFRSNGSRTDFTLKISKLEADDVGVYYCAQNLQLPYTFGGGTKLEIK 拼接正确分析 (46) IPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHS-NGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSSHVPPAFGGGTKLEIKRADAAPTVS Consensus (46) DIQLTQSPASLAVSLGEKVTISCKSS SLL S N YLHWYQQKPGQSPKLLIY ASNL SGVPDRFSGSGSGTDFTLTIS VEAEDLAVYYC Q PYTFGGGTKLEIK

18 Chimeric antibody test Coating 1 st antibody OD Coating 5F8Chimeric (7/16/15) YAL008-1 peptide-bsa 5F8Chimeric (7/30/15) YAL008-1 peptide-bsa YAL008 Mouse NC OD 孙乐等 抗 DKK2 中和活性单克隆抗体及其应用 2014 年在美国申请 ( 第二发明人 )

19 B Epitopes 分析 Score: Non-antigenic: <0; Low: 0-7; High:8-15; Extremely high: >15 Pertuzumab EGFR mab Erbitux HFR LFR CDR Fc HFR LFR CDR Fc HFR LFR CDR Fc N/A N/A N/A Indications Indications Indications Metastatic Breast Cancer? Colorectal Cancer ADA Rates ADA Rates ADA Rates 11/386, 2.8% 49/1001, 5% 1)EGFR mab s variable domain has strong antigenic scores and strong ADA expected to seen in healthy monkeys in pre-clinic study. 2)EGFR mab s ADA rate in human cancer patients will be lower than Erbitux but higher than Pertuzumab. 3)Will be fine for treatment of cancer patients who failed in chemo and radiation therapy.

20 Reduce ADA by Removal of B Epitopes Antigen Epitope Analysis Design New Seq. New Antibodies

21 B Cell Epitopes & 3D 序列总排名 B Epitope B Epitope 排名 3D 相似度 3D 相似度排名 H H H H H H H ) 综合考虑抗原性和 3D 相似度, 我们推荐了 6 条序列, 排序如表中 Ranking 所示.

22 双抗体夹心法检测瞬转表达 选用 5 条改进型的重链分别配合 1 条原始轻链和 5 条改进型的轻链进行瞬时转染, 同时表达原始阿达木作为对照, 结果如下 :

23 抗原包被 ELISA 评价结合力

24 EC50 vs IC50

25 研发误区 4- 亲和力等于抑制力 Binding Activity Inhibition Activity Ab1 Ab2 Ab3 Ab1 Ab2 Ab3 亲和力不等于抑制力

26 干扰 vs 竞争 Infliximab Fab 与 TNFα 结合形成空间位阻从而抑制 TNFR2 结合 TNFα Adalimumab Fab 与 TNFR2 的结合位点有重叠, 在一定程度上竞争抑制 TNFR2 结合 TNFα

27 研发误区 5- 抗原表位确定进入临床 2 期再做 1. 知识产权 2. 毒副作用

28 X 射线晶体学 优点 : 分辨率高, 对生物大分子的分子量无要求 缺点 : 需要制备衍射能力强的单晶样品, 需要处理相位问题 冷冻电镜 优点 : 样品制备简单, 无需高质量单晶, 无相位问题, 对检测样品纯度及浓度要求不高 缺点 : 部分高分辨率的结构解析对硬件要求高

29 氢 / 氘交换质谱 (Hydrogen/Deuterium Exchange Mass Spectrometry, HDX-MS) 骨架酰胺氢在弱氢键或位于蛋白质表面可迅速地交换, 而那些埋藏在内部或涉及稳定氢键则交换的较慢, 通过测量骨架酰胺氢的氢氘交换率可得到蛋白动力学和构象信息 在抗原表位检测中, 通常用游离抗原进行 HDX 实验, 作为抗原 抗体复合物的参比 将两个实验生成的数据进行比较, 具有不同交换动力学的区域被认为是潜在的抗原表位 Hui Wei, et al., Drug Discov Today. 2014

30 氢 / 氘交换质谱 (Hydrogen/Deuterium Exchange Mass Spectrometry, HDX-MS) D 2 O Digestion D 2 O Digestion

31 氢 / 氘交换质谱 (Hydrogen/Deuterium Exchange Mass Spectrometry, HDX-MS) Hui Wei, et al., Drug Discov Today. 2014

32 抑制力恢复 2.5 Inhibition Activity Ab3 Ab2 Ab2-1

33 研发误区 6- 小鼠中抗体药 ADA 数据不可靠 Dilution 1:500 1:1000 1:5000 1: :50000 NC No. 1:500 1:1000 1:5000 1: :50000 NC Pembrolizu mab 5% milk 5% milk +hu IgG 1# # # # # # We have been doing the test incorrectly! 33

34 Mouse ADA Titers vs Human ADA Rates Target Ab Name Type ADA in human Mouse ADA Titer PD-L1 Atezolizumab Engineered full human IgG1 41.5% >1:50K PD-1 Nivolumab Fully human IgG4 12% 1:5K PD-1 Pembrolizumab Engineered humanized IgG4 0.3% 1:1K Anti-Antibody IgG Titers in Mice on Day 14

35 改良型阿达木在小鼠体内免疫原性评价再次验证降低 80% 以上

36 实验方案 给药组别编号供试品剂量动物数量性别 加速组 正常给药组 1-1 抗体药供试品 1-5 雌性 1-2 抗体药供试品 2-5 雌性 1-3 抗体药供试品 3-5 雌性 2-1 抗体药供试品 1 1 mg/kg 10 雌雄各半 2-2 抗体药供试品 2 1mg/kg 10 雌雄各半 2-3 抗体药供试品 3 21mg/kg 10 雌雄各半 加速组 : 给药途径 : 皮下注射 肌肉注射 腹腔注射 取血方式 : 尾静脉取血, 结束后取心脏血 取血时间 : 分别在 D0,D14, D21 取血 正常给药组给药途径 : 皮下给药 (SC) 给药频率 : 每 2 周给药一次, 给药 8 周, 恢复期 4 周 给药剂量 :1mg/kg 取血方式 : 尾静脉取血, 恢复期结束后取心脏血 取血时间 :D0, D14, D28, D42, D56, D70, D84;D85 终放血

37 加速组 Group Mouse No. D14(12/29/2017) D22(1/6/2018) D27(1/11/2018) 1# 1: : : # 1:1000 1: :50000 Ab1 3# 1:5000 1:5000 1: # 1:500 1:5000 1: # 1:1000 1:1000 1: # 1:50000 >1:50000 >1: # >1:50000 >1: :10000 Ab2 3# 1: :10000 >1: # 1: : : # 1:50000 >1:50000 >1: # 1:5000 1: : # 1: :10000 >1:50000 Ab3 3# 1:5000 1: : # 1: :10000 >1: # 1: : :10000

38 组别 采血日期 1# 2# 3# 4# 5# 6# 7# 8# 9# 10# D0 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative D14 1:5000 1:5000 1:5000 1:5000 1:1000 1:5000 1: : :1000 1:5000 D28 1: : : : :5000 1: : : :5000 1:50000 Ab1 D42 1: : : :50000 >1:50000 >1: : : : :50000 D56 >1:50000 >1:50000 >1:50000 >1:50000 >1:50000 >1:50000 >1:50000 >1:50000 >1: :50000 D70 >1:50000 >1:50000 >1:50000 >1:50000 N / A N / A >1:50000 N / A 1:50000 >1:50000 D84 >1:50000 >1:50000 >1:50000 >1:50000 N / A >1:50000 >1:50000 N / A 1:50000 >1:50000 D85( 终 ) >1:50000 >1:50000 >1:50000 >1:50000 N / A >1:50000 >1:50000 N / A 1:50000 >1:50000 D0 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative D14 1: :5000 1:5000 1:50000 >1: : :1000 1: : :10000 D28 1: : :10000 >1:50000 >1:50000 >1: :1000 >1: : :50000 Ab2 D42 1:10000 >1:50000 N / A >1:50000 >1:50000 >1: :5000 >1: : :50000 D56 >1:50000 >1:50000 N / A >1:50000 >1:50000 >1: :10000 >1:50000 >1: :50000 D70 >1:50000 >1:50000 N / A N / A N / A >1: :50000 N / A N / A N / A D84 >1:50000 >1:50000 N / A N / A N / A >1: :50000 N / A N / A N / A D85( 终 ) >1:50000 >1:50000 N / A N / A N / A >1: :50000 N / A N / A N / A Ab3 D0 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative D14 1:100 1:1000 1:100 1: :100 1: :1000 1: :1000 1:50000 D28 1:100 1:1000 1:100 1: :100 >1: :1000 1:10000 N / A 1:50000 D42 1:1000 1: :100 N / A 1:100 >1: :10000 >1:50000 N / A >1:50000 D56 1:5000 1: :100 1: :100 >1: :10000 >1:50000 N / A >1:50000 D70 1:50000 N / A 1:100 1: :100 >1: :50000 >1:50000 N / A >1:50000 D84 1:50000 N / A 1:1000 N / A 1:100 >1: :50000 >1:50000 N / A N / A D85( 终 ) 1:50000 N / A 1:100 N / A 1:100 >1:50000 >1:10000 >1:50000 N / A N / A

39 研发误区 7- 猴子和人不一样, 上临床再说 Study in Healthy Monkeys Low (17.5mg/kg) Rate Day -2 0/8 0% Week 2 3/8 37.5% Week 4 3/8 37.5% Week 6 2/8 25.0% Week 8 2/8 25.0% Week 10 2/8 25.0% Overall 4/8 50.0% Sun L., et al J. Immunol Method, % in Healthy Monkeys vs 43-51% in Healthy Human 39

40 抗体药猴 ADA 阳性血清 Antibody 启动时间 采血时间 血清效价 Human ADA 阿达木类似药 7/24/16 9/27/16 >1: % 美罗华类似药 3/10/16 6/15/16 1: % 爱必妥类似药 3/10/16 6/15/16 1: % 贝伐类似药 3/10/16 6/15/16 1: % 赫赛汀类似药 3/10/16 6/15/16 1: % 抗体药 5/9/17 7/14/17 1: % 抗体药 9/20/17 11/25/17 1:5000 抗体药 11/8/17 2/16/18 1:10000 抗体药 11/8/17 2/16/18 1:5000 抗体药 11/22/17 1/20/18 1: % 抗体药 12/29/17 2/28/18 1:5000 抗体药 1/5/18 2/16/18 1:5000 抗体药 2/1/18 3/1/18 <1:1000 抗体药 2/12/18 3/5/18 1:10000 抗体药 1/11/16 3/28/16 1: %

41 阿达木原研药和改良药在猴子中的免疫原性对比 给药前第 7 天第 14 天第 21 天第 28 天 阿达木原研药 阿达木改良药 F - +/ M F M 阿达木原研药在猴子体内的免疫原性被彻底去除

42 Importance of ADA Test 临床免疫原性分析的意义 Guide the dose in the late stage clinic development ( 将指导抗体新药后期的用药剂量 ) If the immunogenicity is too high, it may pause the development ( 如果影响太强, 或有必要暂停该项目 ) 42

43 研发误区 8- 生物药 ADA 高就是序列上出问题 多聚体导致强免疫原性 43

44 多聚体导致强免疫原性

45 Healthy Subjects vs Patients Humira ADA rates are 43-51% in healthy subjects, 68-75% in Psoriasis(Factor of 1.5), and 38% in MTX-treated RA patients (Factor of 0.8). Roche s anti-pd-l1 ADA rates are 100% in healthy monkeys and 41.5% in cancer patients (Factor of 0.415). ADA data from healthy animals does not equal to observed data from human patients. But the data from healthy animals can be translated by times a factor either bigger than 1.0 for autoimmune diseases or smaller than 1.0 for cancers.

46 Internalization & Cell Killing of Humanized LA22 46

47 Humanized anti-dkk2 antibody 孙乐等 人源化抗 DKK2 中和活性单克隆抗体及其应用 2017 年在美国申请 ( 第二发明人 )

48 Additional Products & Services Catalog Products Mouse anti-humira, anti-avastin mabs,etc Monkey anti-humira, anti-avastin pabs,etc Mouse ant-dm1 MMAE/MMAF mabs DM1-ADC PK assay kits, etc Protein A Assay Kit Services ADA mouse mab and monkey pabs generation ADA assay development for both non-clinic and clinic studies PK assay development HCP assay development Antibody/Antigen Epitope X-ray/H2/H3 exchange study We-chat: Lsun@antibodychina.com Antigenicity prediction, de-immunogenicity and in vivo study 48